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Microsatellite typing is frequently used in disease diagnosis and in genetic association studies. Inter-study consistency and comparability is essential in both applications. In this study, we show that the interlaboratory comparison of microsatellite sizes is often discrepant and misleading. This is a matter of great concern in the recent literature. However, accurate allele designation is easily attainable by the simple procedures we report, which are applicable to all gel-based genotyping methods. These involve: 1) the creation of dedicated standards for a specific microsatellite by PCR-amplifying representative alleles to generate an allelic ladder with comparable electrophoretic characteristics; 2) including both internal and external standards during electrophoresis to facilitate alignment. In addition, we recommend procedures that will improve inter-study comparability of all microsatellite analyses regardless of genotyping method. These involve: 1) cloning and sequencing representative microsatellite alleles to obtain accurate size designation; 2) sharing alleles of known sequence between laboratories to use as standards. We report on the typing of natural resistance-associated macrophage protein ( NRAMP2 ) and interferon-γ ( IFNG ) gene microsatellites as examples, the latter of which is crucial to many pathogenic processes. We describe in detail the varying allele sizes obtained by different methods, which prevent meaningful inter-study comparisons.

clinical chemistry and laboratory medicine

Accurate Microsatellite Typing and Inter-study Comparison: Pitfalls and Solutions Using Interferon-γ (IFNG) and Natural Resistance-associated Mocrophage Protein 2 (NRAMP2) Genes as Examples